Hepatoprotective and Metabolic Restorative Potential of a Polyherbal Juice Formulation in Non Alcoholic Fatty Liver Disease

Abstract

One of the most common chronic liver disorders worldwide, non-alcoholic fatty liver disease (NAFLD), is strongly linked to metabolic diseases such type 2 diabetes, obesity, insulin resistance, and dyslipidaemia. It is characterised by an excessive buildup of fat in hepatocytes in people who drink very little or nothing. Cirrhosis, fibrosis, simple steatosis, and nonalcoholic steatohepatitis (NASH) are some of the phases of the disease's evolution that might eventually result in hepatocellular cancer or liver failure. The primary care approach for NAFLD is still lifestyle modification because there isn't yet a single authorised pharmaceutical medication. There is growing interest in studying both conventional and herbal medicinal treatments to treat and prevent this illness.Using medicinal plants including Curcuma longa, Cuminum cyminum, Foeniculum vulgare, Trachyspermum ammi, Phyllanthus emblica, and Piper nigrum, the current work focuses on creating a polyherbal juice composition based on Ayurvedic principles. Numerous bioactive phytoconstituents, such as flavonoids, phenolic compounds, alkaloids, terpenoids, tannins, and essential oils, are known to be present in these plants. These components have important pharmacological actions, including hepatoprotective, antioxidant, anti-inflammatory, hypolipidemic, and digestive stimulating qualities, which may help lower the inflammation, oxidative stress, and lipid buildup linked to NAFLD.To maximize the phytochemical yield, the herbal components were extracted utilizing appropriate techniques such decoction and maceration. To find significant bioactive components in the extracts, preliminary phytochemical screening was done. To guarantee the quality, safety, and stability of the polyherbal juice formulation, preformulation investigations such as physicochemical evaluation, solubility analysis, viscosity determination, stability testing, and microbiological studies were carried out.The study emphasizes the possibility of polyherbal formulations as an all-natural and effective treatment strategy for NAFLD management. To confirm the new formulation's effectiveness, safety, and treatment mechanisms, more experimental and.clinical research is necessary

Keywords

Hepatoprotective , Metabolic Restorative Potential, Polyherbal Juice Formulation, Non Alcoholic Fatty Liver Disease

Introduction

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

A combination extraction technique used in pharmacognosy to maximise the yield of active ingredients from plant materials is maceration with water following decoction. In order to extract water-soluble chemicals, the coarsely ground plant material is first put through a procedure called decoction, which involves boiling it in water for a predetermined amount of time. The maceration phase involves filtering the decoction and then soaking the leftover plant residue (marc) in new water at room temperature. To allow the solvent to permeate the plant tissue and dissolve any remaining soluble components, the mixture is left for several hours with sporadic stirring. To increase the total extraction effectiveness, the filtered macerated liquid can be mixed with the decoction extract. The highest possible recovery of phytochemicals from the plant material is ensured by this technique. Following cooling, the decoction was put in a sealed container and left to macerate at room temperature for 24 to 48 hours. To enhance the phytoconstituent extraction, the contents were periodically agitated. After that, Whatman filter paper and muslin cloth were used to filter the extract. [132, 133].

Phytochemical Screening Herbal Extracts:

 

Phytochemical Screening of Curcima Longa L.:

Several phytoconstituents found in the powdered crude medication have been identified using qualitative chemical testing as part of the chemical evaluation. Several researchers conducted preliminary phytochemical analyses of the Curcuma longa rhizome's utilizing commonly used precipitation and coloration procedures to extract aqueous, acetone, ethanolic, chloroform, and methanolic substances. According to multiple researchers, these analyses showed the presence of carbohydrates, proteins, alkaloids, glycosides, terpenes, steroids, flavonoids, tannins, and saponins [104,105,106].

Preparation of the Extract

The rhizomes of Curcuma longa were collected and sun dried, cut into small pieces The small piece of dried rhizome was then grinded to get a fine powder, which is ready for use [105,106].

1. Alkaloid test

After combining the extract with three millilitres of diluted hydrochloric acid, it was thoroughly filtered. The filtrate was thoroughly examined using the subsequent test [106]:

 (a) Mayer's Test :A few drops of Mayer's reagent are added by the test tube's side to one or two millilitres of filtrate. The presence of alkaloids was detected by a white or creamy precipitate [104, 107, 105, 108]

(b) Wagner Test: Wagner's reagent was added to 1 or 2 millilitres of the filtrate extract; the production of a brown, reddish precipitate indicates the presence of alkaloids [104, 107, 105].

(c) Dragendroff’s Test: The presence of alkaloids is indicated by the production of a noticeable yellow precipitate when 1-2 millilitres of Dragendorff's reagent are added to a few millilitres of filtrate [107, 105].

2. Glycoside Testing

(a) Adding equal amounts of Fehling's solutions A and B to a 2 ml test solution and heating the mixture yields a positive glycoside result. There was a brick-red precipitate [105].

(b) Legal's Test: Add pyridine and alkaline sodium nitroprusside to a 2 ml or 1 ml test solution; the presence of glycoside is indicated by a blood red or pink colour [104,105,106].

(c) Keller-Killani Test: Add a drop of FeCl3 treated with extract to two millilitres of glacial acetic acid.Glycoside is present when a brown ring forms [104, 106].

3. Flavonoids Test:

(a) Shinoda Test: Concentrated H2SO4 was added dropwise to a 2 ml test solution containing a few pieces of magnesium ribbon. The results shows pink scarlet or crimson red colour [104, 105].

(b) Alkaline Reagent Test: A sodium hydroxide solution was added to the test solution, giving it a yellow or red hue [104, 105].

4. Tannins Test:

1. Ferric Chloride Test: Add drops of ferric chloride solution to the extract solution. Gallic tannins were found to be blue, while catecholic tannins were found to be green-black [104, 107, 105].

(b) Gelatin Test: By combining two millilitres of test solution with a 1% Gelatin solution that contains ten percent sodium chloride, a white precipitate is produced [105].

5.Saponins Test :

Foam Test: The following is how researchers attempt to determine whether saponins are present: After shaking 5 ml of extract with 20 ml of distilled water, the mixture was brought to a boil. Saponins can be detected by frothing [104,107].

Phytochemical Screening of Cuminum Cyminum L. :

Screening for Phytochemicals

The Madurai local market is where the cumin seeds were bought. Using established methods, the methanolic extracts were put through chemical testing to identify several phytoconstituents [109].
Phenol Test (FeCl3 Test)

Three millilitres of distilled water and a few drops of a 10% aqueous ferric chloride solution were added to one millilitre of the extract. Phenols are present when a blue or green hue forms.
Flavonoid Test (Shinoda Test)

One millilitre of a 1% ammonia solution was added to two millilitres of the extract. The presence of flavonoids is indicated by a yellow appearance.

Tannin Test (FeCl3 Test)

One millilitre of 0.008 M potassium ferricyanide and one millilitre of 0.02 M ferric chloride with 0.1 N HCl were added to one millilitre of the extract. The presence of tannins is indicated by a blue-black appearance.

Alkaloids Test (Wagner's Reagent Test)

Two millilitres of Wagner's reagent were combined with about one millilitre of crude extract. Alkaloids are indicated by a reddish-brown precipitate.

Test for Carbohydrates (Benedict's and Fehling's tests)

Fehling's test

Two millilitres of crude extract were added to an equal volume of Fehling A and Fehling B reagents, which were then gently heated. Reducing sugars are present when a brick-red precipitate forms at the bottom of the test tube.
Benedict's examination

Benedict's reagent (2 ml) and crude extract (1 ml) were combined and heated. The presence of the carbohydrates was indicated by the formation of a reddish brown precipitate.

Protein Testing (Millon's Test, Ninhydrin Test)

Millon's examination

When 1 millilitre of crude extract and 2 millilitres of Millon's reagent were combined, a white precipitate formed that became red when heated gently, indicating the presence of protein.
Cardiac glycoside test (Keller-Kiliani test)

Two millilitres of glacial acetic acid with one drop of ferric chloride solution were added to five millilitres of extract. One millilitre of pure sulphuric acid was placed underneath this. The deoxy-sugar feature of carotenoids is indicated by a browning of the interface. A greenish ring may develop gradually over the thin layer of acetic acid, while a violet ring may emerge beneath the brown ring.

Saponin Test (Foam Test)

In a test tube, two millilitres of crude extract and five millilitres of distilled water were combined and forcefully shaken. Add a few drops of olive oil. Stable foam formation was interpreted as a sign that saponins were present.
Coumarin Test (Sodium Hydroxide Test)

The extract was mixed with 10% sodium hydroxide and chloroform. The development of a yellow hue indicates the presence of coumarin.

Terpenoids Test (Salkowski test)

To create a layer, 5 ml of extract, 2 ml of chloroform, and 3 ml of strong sulphuric acid were carefully added. The formation of a reddish-brown interfacial colouration suggests the presence of terpenoids.
Test for Steroids (Salkowski Test)

0.5 ml of crude extract containing 2 ml of sulphuric acid was mixed with 2 ml of acetic anhydride. The presence of steroids is indicated by the colour of the samples changing from violet to blue or green.
The sodium hydroxide test is used to detect quinones.
One millilitre of crude extract was mixed with diluted sodium hydroxide. Quinones are indicated by a blue-green or red hue.

Anthraquinone test (Borntragers test)

10% hydrochloric acid and 0.5 g of extract were heated in a water bath for a few minutes. After filtering, it was left to cool. The filtrate was mixed with an equal volume of CHCl3. The mixture was heated after a few drops of 10% ammonia were added. The development of a rose-pink hue signifies the presence of anthroquinones in n-hexane, chloroform, ethyl acetate, and methanol.
Analysis of Fourier Transform Infrared Spectroscopy (FTIR)

An Agilent Cary 630 FTIR spectrometer with Micro-lab PC software and an ATR sampling device with a resolution of 8 cm-1 and a scan range of 4000 cm-1 to 650 cm-1 was used to analyse the extracts.[110, 111]

Phytochemical Screening of Foeniculum vulgare:

Analysis of phytochemicals Using the corresponding calibration curve that was generated calorimetrically, phytochemical analysis was performed in hexane, chloroform, and methanol extracts of fennel seeds in terms of total phenolic content, total flavonoid content, and total tannin content.

Total phenolic content:

Chandrasekara and Shahidi slightly modified the Singleton and Rossi20 method to assess the total phenolic (TPC) content in hexane, choloroform, and methanolic extracts of fennel seeds [112]. In a centrifuge tube, precisely 0.5 mL of fennel seed extract and 0.5 mL of Folin Ciocalteu Reagent reagent were combined. Each test tube was filled with 1 mL of saturated Na2CO3 solution to neutralise the reaction, and the total volume was increased to 10 mL using distilled water. For 35 minutes, the mixture was incubated in the centrifuge tubes at 24°C in the dark. The mixture was then centrifuged for ten minutes at 4000 rpm, and the absorbance was measured at 725 nm in

comparison to the reagent blank.Gallic acid equivalents (mg GAEg-1) were used to record the data, and the TPC was calculated by creating a standard curve utilising various gallic acid concentrations.

Total flavonoid content :

The method used by Kim et al. [113] was modified by Chandrasekara and Shahidi [114] to determine the total flavonoid content (TFC). 4 mL of purified water and 1 mL of fennel extract were combined. The centrifuge tubes were then filled with 300 μL of NaNO2 and left to incubate for five minutes. After adding 300 μL of AlCl3 to the reaction mixture and letting it stand for a minute, 2 mL of 1M NaOH and 2.4 mL of distilled water were added. These solutions were immediately mixed and then incubated at 24°C for 15 minutes in the dark. After centrifuging the mixture for five minutes at 4000 rpm, the absorbance at 510 nm was measured. The TFC was expressed in milligrams of catechin equivalents (mg CAEg-1).

.Total tannins content:

The Sadasivam and Manickam method was used to determine total tannins [26]. After precisely 1 mL of extract was combined with 6.6 mL of water, 0.5 mL of Folin-Denis reagent and 1 mL of Na2CO3 solution were added, and the volume was increased to 10 mL with purified water. After 30 minutes of room temperature incubation, the test mixture's absorbance at 700 nm was measured in comparison to the reagent blank. Tannic acid (10–100 μg) was used to compute the amount of tannin from the standard curve. The standard was expressed as mg tannic acid equivalents (mg/TAE/g).[115]

Phytochemical Screening of Trachyspermum Ammi:

Flavonoid testing:

Alkaline Reagent Test: The crude extract was mixed with 2 millilitres of 2.0 percent NaOH. When two drops of diluted acid were added, the yellow tinge that indicated the presence of flavonoids vanished to colourlessness.[116]

Check for tannins:

Ferric chloride test: A few drops of a 10% ferric chloride solution were added to two millilitres of extract. A blackish blue tint appeared when gallic tannins were present, while a green-blackish colour appeared when catechol tannins were present.[117]

Check for alkaloids.

Mayer's test: Add 1 mL of Mayer's reagent to 1 mL of extract. A white, yellow, or cream-coloured precipitate indicates the presence of alkaloids [.118].

Check for saponins.

Foam test: A graduated cylinder was filled with 1 mL of extract solution diluted to 20 mL with distilled water and shaken for 15 minutes. The development of foam indicates the presence of saponins.[119].

Check for glycosides.

Keller-Kiliani test: Two millilitres of glacial acetic acid containing one or two drops of a 2% FeCl3 solution were combined with the crude extract. The extract was then transferred into a second test tube that included two millilitres of concentrated H2SO4. The development of a brown ring at the contact indicated the presence of cardiac glycosides.[120]

Phyllanths emblica Phytochemical Screening:
The ethanolic and aqueous extracts of Emblica officinalis were analysed using a conventional approach in the study by Jeevalatha et al. (2022) [121] to identify the presence of several phytoconstituents, such as alkaloids, tannins, glycosides, flavonoids, and saponins.

Check for Phenol :

Test for ferric chloride: One millilitre of the extract was mixed with three millilitres of distilled water and a few drops of a 10% aqueous ferric chloride solution. The presence of phenols is indicated by the formation of a green tint.

Check for Flavonoids :

Shinoda examination: One millilitre of a 1% ammonia solution was added to two millilitres of the extract. The presence of flavonoids is indicated by their yellow appearance.

Check for Tannins:

Ferric chloride test: Two millilitres of the extract were mixed with one millilitre (1%) of a 1% ammonia solution. The hue yellow indicates the presence of flavonoids.

Check for Alkaloids :

Mayer's examination :One millilitre of the extract, one millilitre of 0.02 M ferric chloride containing 0.1 N HCl, and one millilitre of 0.008 M potassium ferricyanide were mixed together. A blue-black appearance indicates the presence of tannins.

Check for Carbohydrates :

Fehling's test: Equal quantities of Fehling A and Fehling B reagents were mixed and heated gradually before adding two millilitres of crude extract. Reducing sugars are present when a brick-red precipitate forms on the bottom of the test tube.

Check for Proteins

a) The Millon test: One millilitre of crude extract and two millilitres of Millon's reagent were mixed to create a white precipitate that became scarlet when heated slowly, signifying the presence of protein.
b) The ninhydrin test :One millilitre of crude extract and two millilitres of a 0.2% ninhydrin solution were combined and boiled. A violet-colored precipitate indicated the presence of proteins and amino acids.
Check for Glycosides
a) Sodium hydroxide test The extract was treated with 1 millilitre of sodium hydroxide and 1 millilitre of water. Glycosides are indicated by the appearance of a yellow tint.

b)The cardiac glycoside Keller-Kiliani test: Five millilitres of extract were mixed with two millilitres of glacial acetic acid and one drop of ferric chloride solution. One millilitre of sulphuric acid concentration was added to this. A browning of the interface indicates the presence of a deoxy sugar property in cardenolides. A violet ring may appear beneath the brown ring, and a greenish ring may develop very slowly within the thin coating of acetic acid.

Check for Terpenoids

Salkowski examination:To make a layer, five millilitres of extract and two millilitres of chloroform were carefully mixed with three millilitres of strong sulphuric acid. The reddish-brown hue of the interface indicates the presence of terpenoids.

Check for Coumarin

The extract was mixed with 10% sodium hydroxide and chloroform to test for coumarins. The development of a yellow hue indicates the presence of coumarin.

Check for Saponins

Test of foam: Two millilitres of crude extract and five millilitres of distilled water were mixed together in a test tube and forcefully stirred. Add a few drips of olive oil. The creation of stable foam was thought to be a sign that saponins were present.

Check for Steroids

Salkowski examination: Two millilitres of sulphuric acid and two millilitres of acetic anhydride were combined with 0.5 millilitres of crude extract. Steroids are present when a sample's colour changes from violet to blue or green.

Check for Quinones

Quinone examination Diluted sodium hydroxide was mixed with one millilitre of crude extract. Red or blue-green hues are indicative of quinones.

Check for Anthraquinones
The Borntrager test:Each extract weighed 0.5 g and was briefly boiled in a water bath containing 10% hydrochloric acid. It was filtered and then allowed to cool. The filtrate was treated with the same volume of CHCl3. The mixture was heated after a few drops of 10% ammonia were added. When a rose-pink hue appears, anthraquinones are present.
Gallic acid was measured using High Performance Thin Layer Chromatography (HPTLC).[122]

Phytochemical Screening test of Piper Nigrum:

Check for saponins :
In a water bath, two grams of the powered sample were cooked in twenty millilitres of distilled water. To create a stable, long-lasting froth, ten millilitres of the filterable were combined with five millilitres of distilled water and forcefully shaken. Three drops of olive oil were added to the mixture, and it was vigorously shaken. then watched to see if an emulsion formed.

Check for tannins:

After boiling 0.5 g of the dried powdered sample in 20 ml of water, it was filtered. After adding a few drops of 0.1% ferric chloride, the colour was checked for brownish or bluish black.

Alkaloids test (Meyer's test):

After boiling 0.5 g of the dried powdered sample in 20 ml of water, it was filtered. Meyer's reagent was applied by t1 side of the test tube to a few drops of the filtrate. The test is positive if there is a creamy or white precipitate.

Check for flavonoids:

After heating a portion of the powdered sample with 10 millilitres of ethyl acetate over a steam bath for three minutes, the mixture was filtered. Ammonia that had been diluted was mixed with 4 millilitres of the filtrate. The presence of flavonoids is indicated by yellow colouring.

Check for cardiac-related glycosides:

One drop of 1% FeCl3 and two millilitres of glacial acetic acid were used to dissolve around 0.5 g of the extract. Concentrated H2SO4 was used to underlay this. The presence of a deoxy sugar, which is typical of cardiac glycosides, was shown by a brown ring that was produced at the interface. In the acetic acid layer, a greenish ring may form just above the ring and progressively expand throughout the layer, or a violet ring may appear underneath the ring.

Look for phenolic compounds.
Ferric chloride test: Five millilitres of distilled water were diluted with three hundred milligrams of extract, then filtered. Five percent ferric chloride was added to the filtrate. Phenolic substances are indicated by a dark green hue.[123]

Preformulation Studies of PolyHerbal Juice Formulation:

Physicochemical parameters (124–127): The herbal syrup was assessed for a number of physicochemical characteristics, including pH, Wt/ml, Specific Gravity, and physical appearance (colour, aroma, and taste).
a) Colour analysis: Five millilitres of the finished syrup were put into watch glasses and exposed to a white tube light on a white background. The colour was seen with the unaided eye.
c) Odour analysis: Two millilitres of the finished syrup were smelt separately. To counteract the impact of prior smelling, a two-minute gap was maintained between the two scents.
c) Taste test: A small amount of the finished syrup was taken and its flavour was assessed using the tongue's taste buds.

d) Determining pH: - Fill a 100 ml volumetric flask with precisely measured 10 ml of the finished syrup, then top it up with 100 ml of distilled water. For roughly ten minutes, the solution was sonicated. A digital pH meter was used to measure the pH.

e) Specific gravity at 250C: A completely dry and clean Pycnometer was chosen, and its contents were weighed after it was filled with freshly boiled and cooled water at 250C. Assuming that one millilitre of water at 250 degrees Celsius weighs 0.99602 grams in air with a density of 0.0012 grams per millilitre. The Pycnometer's capacity was computed. Adjusting the temperature of the final syrup to about 200C and the Pycnometer was filled with it. Then the temperature of the filled Pycnometer was adjusted to 250C, any excess syrup was removed and weight was taken.

Stability testing:

Nine portions of the final syrup (1A, 1B, 1C, 2A, 2B, 2C, 3A, 3B, and 3C) were taken in amber-colored glass bottles and kept at accelerated temperatures at 40C, room temperature, and 470C, respectively. The samples were tested for all the physicochemical parameters, turbidity, and homogeneity at intervals of 24, 48, and 72 hours to look for any changes.

Microbiological studies

Prepared juice blends were studies for microbial load. The microbial content of all the samples was estimated by using total plate count technique (APHA, 1967).[128]

Solubility Analysis:

An essential component of preformulation research for polyherbal juice compositions is solubility analysis. It is done to find out how soluble herbal components are in various solvents, including water, ethanol, and methanol. The physicochemical characteristics of the phytoconstituents in the formulation are better understood thanks to this investigation. The juice's active ingredients are distributed uniformly when they are properly soluble. It also aids in the selection of appropriate solvents for formulation development and extraction. To see how the polyherbal extract dissolves, it is combined with various solvents and agitated for a predetermined amount of time. The amount of dissolved components is then ascertained by filtering and analysing the mixture. The stability and bioavailability of the herbal formulation are improved by good solubility. Additionally, it enhances the polyherbal juice's medicinal efficacy. Solubility study is therefore essential to the creation of stable and potent herbal formulations.[130]

Rheology /Viscocity:

Using the equation, the apparent viscosity was used to calculate the amount of time needed for each sample to pass through a 10 ml pipette.

Flow rate is equal to pipette volume (ml) divided by flow rate (seconds) [129].

Preservative Compatibility:

A study on preservative compatibility is a crucial aspect of the preformulation analysis for polyherbal juice. This evaluation focuses on the interaction between the preservatives and the herbal ingredients in the formulation. Conducting this study ensures that the chosen preservative does not compromise the stability, efficacy, or safety of the herbal components. Commonly used preservatives, such as sodium benzoate or potassium sorbate, are incorporated into the polyherbal juice, and any physical or chemical alterations are monitored. The formulation is assessed for various parameters, including color alterations, precipitation, pH changes, and microbial growth over time. A suitable preservative effectively prevents microbial contamination and enhances the shelf life of the polyherbal juice. Thus, testing for preservative compatibility is vital for preserving the quality, stability, and safety of herbal formulations.

CONCLUSION

Non-alcoholic fatty liver disease (NAFLD), one of the most common chronic liver disorders in the world today, is closely linked to metabolic issues such obesity, dyslipidemia, and insulin resistance. Simple steatosis develops into non-alcoholic steatohepatitis (NASH), fibrosis, and eventually cirrhosis or hepatocellular carcinoma if treatment is not received. The cornerstone of treatment for NAFLD is lifestyle changes like dietary restriction and increased physical exercise, as there is presently no approved medication for the condition. Alternative therapy choices, especially herbal therapies, are becoming more and more popular due to their natural origins, therapeutic benefits, and relatively reduced side effects.

The potential use of a polyherbal formulation that includes therapeutic plants including Curcuma longa, Cuminum cyminum, Foeniculum vulgare, Trachyspermum ammi, Phyllanthus emblica, and Piper nigrum is highlighted in this review. Flavonoids, phenolic compounds, alkaloids, tannins, terpenoids, and essential oils with hepatoprotective, anti-inflammatory, antioxidant, hypolipidemic, and digestive stimulating qualities are among the many bioactive phytoconstituents found in these plants. Because oxidative stress, lipid buildup, and inflammation are important factors in the pathophysiology of NAFLD, these pharmacological actions are especially pertinent to its management.According to the Ayurvedic idea of polyherbalism, mixing several medicinal herbs may have a synergistic therapeutic impact that increases efficacy while lowering dosages and possible side effects. Decoction and maceration are two preparation techniques that aid in the extraction of water-soluble active components, improving the availability of phytochemicals. Preformulation research and phytochemical screening are other crucial procedures for assessing the herbal formulation's stability, safety, and quality.Overall, the results point to a potential natural strategy for promoting liver function and controlling NAFLD: a polyherbal juice formulation including several therapeutic herbs. However, before it can be suggested for broad therapeutic usage, more experimental research and clinical trials are required to scientifically establish its efficacy, safety, and mechanism of action.

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